Standard nucleic acid separation techniques limit researchers' abilities to analyze samples for nucleic acids that are present in low abundance, such as mutations. In particular, it is difficult to resolve rare nucleic acids that are present at low concentrations in the presence of closely-related nucleic acids, e.g., wild-type DNA.
In order to resolve rare mutations in a sample, all of the nucleic acids in a sample are typically amplified prior to isolation and analysis. For example, using Polymerase Chain Reaction (PCR) amplification, each nucleic acid in a sample can be amplified one million times (or more). Theoretically, there will be a million-fold increase of each nucleic acid originally present, and, thus, a greater opportunity to isolate and find the nucleic acids in low abundance. In practice, however, PCR amplification has significant drawbacks when used to amplify nucleic acids that are present in low abundance. The PCR reaction is stochastic, and to the extent that a low-abundance nucleic acid is not amplified in the first few rounds of PCR, it likely will not be detected. In addition, PCR amplification introduces sequence errors in the amplicons because of the lack of fidelity in the polymerases used. In some systems, PCR and/or sequencing errors can result in error rates on the order of 0.1% or higher. If the error rate is greater than or equivalent to the mutation rate in a system, it is difficult to reliably detect the mutations in the system.
An additional shortcoming of PCR amplification is that at least a portion of the sequence of interest must be known prior to the amplification. During the amplification process, a primer having a sequence at least partially complementary to the target is used to begin amplicon construction. The primer need not be exactly complementary to the mutation, but it must be similar enough to prompt a polymerase to copy the region of interest. When a known mutation is sought, the sequence of the corresponding primer is easily determined, and nucleic acids can be reliably amplified, subject to the shortcoming discussed above. When an unknown mutation is present, however, it is possible that the selected primers will not hybridize with the unknown mutation. If the primers do not hybridize with the unknown mutation, the mutation will not be amplified, and it is very unlikely that the unknown mutation will be later identified in the presence of millions-fold excesses of known mutations. Furthermore, if the unknown mutation is only a matter of one or two base changes, it is impossible to distinguish the mutation from an error introduced by the amplification process itself.
There is a need for techniques that easily separate closely-related mutations without introducing errors into the sample. There is also a need for techniques to isolate unknown mutations in a nucleic acid sample.